PBMCs were isolated from whole blood by Ficoll-Paque density gradient centrifugation (GE Healthcare Bio-sciences), as described previously (55 (link)). CD8+ T cells were subsequently isolated from PBMCs by negative selection using the EasySep Human CD8+ T Cell Enrichment Kit (STEMCELL Technologies Inc.) according to the manufacturer’s instructions. The CD8+ T cells were maintained in RPMI-1640 medium supplemented with 10% human serum, 200 u/ml penicillin, 200 µg/ml streptomycin, 1 mM L-glutamine, and 10 mM HEPES (55 (link)). Cells were activated for 72–96 h in a cell culture dish coated with 10 µg/ml mouse anti-human CD3 antibody (Biolegend) and 10 µg/ml mouse anti-human CD28 antibody (Biolegend).
Mouse anti human cd28 antibody
Manufactured by BioLegend
The Mouse anti-human CD28 antibody is a monoclonal antibody that binds to the CD28 cell surface receptor on human T cells. CD28 is a co-stimulatory molecule that plays a critical role in T cell activation and proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti human cd3 antibody, by BioLegend (2 mentions)
Ficoll paque, by GE Healthcare (2 mentions)
Easysep human cd8 t cell enrichment kit, by STEMCELL (2 mentions)
Dynabeads m 270 epoxy, by Thermo Fisher Scientific (2 mentions)
Mouse anti human cd3 antibody clone ucht1, by BioLegend (2 mentions)
Mouse igg1 isotype antibodies, by R&D Systems (2 mentions)
Rosettesep human t cell enrichment cocktail, by STEMCELL (2 mentions)
4 protocols using mouse anti human cd28 antibody
1
CD8+ T Cell Isolation and Activation
2
Isolation and Activation of Primary T Cells
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Jurkat T cells were obtained from the ATCC and maintained in RPMI medium supplemented with 10% FBS and 1% penicillin and streptomycin. Peripheral blood was acquired from the New York Blood Center. Total CD3+ T cells were isolated by density gradient centrifugation (Lymphoprep) and adverse selection using the RosetteSep human T cell enrichment cocktail (Stemcell). Primary T cells were directly employed in stimulation assays or maintained in culture. T cell cultures were maintained in complete RPMI, containing 10% FCS, MEM nonessential amino acids, 1 mM sodium pyruvate, 100 IU/ml of penicillin, 100 μg/ml streptomycin, and GlutaMAX-I. For stimulation, Dynabeads M270-Epoxy (Thermo) was covalently conjugated with combinations of mouse anti-human CD3 antibody (clone UCHT1, BioLegend), mouse anti-human CD28 antibody (BioLegend), recombinant human PD-L1 human IgG1 Fc chimera protein (R&D Systems), or mouse IgG1 isotype antibodies (R&D Systems) following the manufacturer’s recommendations. All Jurkat and primary T cell stimulations were performed with beads at a 1:5 cell-to-bead ratio.
3
PBMC Isolation and CD8+ T Cell Activation
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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll-Paque density gradient centrifugation (GE Healthcare Bio-Sciences), as described previously (Chimote et al., 2018 (link)). CD8+ T cells were isolated from PBMCs by negative selection using the EasySep Human CD8+ T Cell Enrichment Kit (STEMCELL Technologies Inc.). The CD8+ T cells were maintained in RPMI 1640 medium supplemented with 10% human serum, penicillin (200 U/ml), streptomycin (200 μg/ml), 1 mM L-glutamine, and 10 mM Hepes. For all experiments, cells were activated in a cell culture dish pre-coated with mouse anti-human CD3 antibody (10 μg/ml) (BioLegend) and mouse anti-human CD28 antibody (10 μg/ml) (BioLegend) for 72 to 96 h, except where specified.
4
Isolation and Stimulation of Primary T Cells
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